CN107488710A - A kind of purposes of Cas albumen and the detection method and kit of target nucleic acids molecule - Google Patents

A kind of purposes of Cas albumen and the detection method and kit of target nucleic acids molecule Download PDF

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CN107488710A
CN107488710A CN201710573752.0A CN201710573752A CN107488710A CN 107488710 A CN107488710 A CN 107488710A CN 201710573752 A CN201710573752 A CN 201710573752A CN 107488710 A CN107488710 A CN 107488710A
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dna
cas12a
target
nucleic acids
target nucleic
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CN107488710B (en
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成秋香
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Shanghai Tolo Biotechnology Co ltd
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Shanghai Tolo Biological Technology Co Ltd
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Priority to EP18832456.0A priority patent/EP3653722A4/en
Priority to CN202410308071.1A priority patent/CN118207297A/en
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Priority to JP2020523474A priority patent/JP2020530779A/en
Priority to SG11202000336RA priority patent/SG11202000336RA/en
Priority to KR1020207003220A priority patent/KR20200035956A/en
Priority to AU2018299445A priority patent/AU2018299445B2/en
Priority to EA202090290A priority patent/EA202090290A1/en
Priority to PCT/CN2018/082769 priority patent/WO2019011022A1/en
Priority to US16/631,157 priority patent/US20230002811A1/en
Priority to MX2020000481A priority patent/MX2020000481A/en
Priority to CN201880046701.5A priority patent/CN111094588B/en
Priority to NZ760987A priority patent/NZ760987B2/en
Priority to CA3069788A priority patent/CA3069788A1/en
Priority to MA049579A priority patent/MA49579A/en
Priority to IL272005A priority patent/IL272005A/en
Priority to PH12020500103A priority patent/PH12020500103A1/en
Priority to ZA2020/00682A priority patent/ZA202000682B/en
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Abstract

The invention provides a kind of purposes of Cas albumen and the detection method and kit of target nucleic acids molecule, the detection method of target nucleic acids molecule includes adding guide RNA, Cas12a, nucleic acid probe into the reaction system containing target nucleic acids molecule to be checked, and it is detected after the completion of reaction.Whether the method using the present invention can contain target nucleic acid sequence in quick detection sample.By and nucleic acid amplification technologies combination, the sensitivity of the detection method can be greatly improved.According to the characteristics of this method, it is named as HOLMES (one HOur Low cost Multiplex Efficient Simple assay), i.e. one hour, low cost, multichannel, efficient, simple analysis method, this method can be used for quick detection pathogenic microorganism, gene mutation and special target DNA etc..

Description

A kind of purposes of Cas albumen and the detection method and kit of target nucleic acids molecule
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to a kind of for target nucleic acids Molecular Detection Method.
Background technology
Specific nucleic acid detecting molecule (Nucleic acid detection) method has important application value, such as The detection of pathogen, hereditary disease detection etc..In terms of pathogen detection, because every kind of pathogen microorganism has its only nothing Two feature nucleic acid molecule sequence, therefore the nucleic acid molecules detection for particular species, also referred to as diagnostic nucleic acid can be developed (NADs, nucleic acid diagnostics), in food security, environmental microorganism pollution detection, human body pathogenic bacterial infection It is significant (O'Connor and Glynn 2010, Scheler, Glynn et al.2014) Deng field.Another Aspect is to the SNP of people or other species (SNPs, single nucleotide polymorphisms) Detection.Understand that the relation between hereditary variation and biological function provides for modern molecular biology up in genomic level New Century Planned Textbook, and wherein SNPs is closely related to the function of biology, evolution and disease etc., therefore SNPs detection and analysis skill The development of art is particularly important.
The method for having established many NADs at present, primarily directed to the detection of specific DNA molecule, also there is Part Methods For RNA molecule.As a rule, DNA molecular is highly stable, therefore detection sample can derive from the biological sample of a series of complex This；And RNA is then very easy to degraded, therefore extreme care is needed during processing.In the seventies in last century, establish in restricted The method of enzyme cutting digestion detection, carried out specific inspection later and the methods of having developed Southern, Northern and dot hybridization Survey nucleic acid molecules detection.1985, after PCR method turns into normal experiment method, it result in that molecular biology is exponential to be entered Step.The specific nucleic acid molecules detection established at present usually requires to be divided into two steps, and the first step is the amplification of purpose nucleic acid, and second Step is purpose nucleic acid detection.Round pcr is foundation at first and amplification method the most frequently used at present, at present on PCR method basis On, the probe of fluorescence labeling is introduced, the amplification situation of target, referred to as Realtime PCR can be detected in real time.Realtime PCR is not only quick, highly sensitive detection method, while this method can also carry out quantitative analysis.Except PCR amplification side Method, many alternatives, such as ligase chain reaction, branched DNA are also set up amplification,NASBA,SDA,transcription-mediated amplification,loop mediated amplification,rolling circle amplification and TwistAmpTMDeng.These many alternatives Advantage is isothermal, that is to say, that only needs a temperature to complete to react, is followed without the heat as PCR Ring instrument.The method of detection of nucleic acids is in addition to Realtime PCR can be done directly amplification and detection, FISH hybridization techniques (Fluorescence in situ hybridization) is the most frequently used detection method, in situ by labelled molecular probes Hybridize with the target sequence of complementation.In addition, biosensors, nanopores and next- are also developed at present The detection methods such as generation sequencing technologies, but these methods usually require expensive experiment and set It is standby.
Detection to SNPs needs also exist for into amplification the methods of performing PCR first, so as to obtain enough areas containing SNP site Domain fragment is further detected.More commonly used method has：Primer extend (primer extension), hybridization (hybridization) (ligation) and digestion (enzymatic cleavage), are connected.After the above method is completed, Need to carry out specific method detection, such as Mass Spectrometer Method, fluoroscopic examination, chemiluminescence detection etc..
Although detection of nucleic acids have developed many detection methods as described above, in some cases, how more It is important developing direction to accelerate fast, easy, cheap detection, such as pathogen quick detection in the wild, medicaments insensitive SNP quick detections etc..In 2016, Collins etc. was based on CRISPR-Cas9 specific recognitions and cuts the spy of target sequence Point, the method for developing quick cheap detection zika virus (Zika).2017, cutting edge of a knife or a sword etc. had using CRISPR-Cas13a The method that the characteristics of " alternative activity " (collateral effect) establishes Rapid nucleic acid detection, i.e. Cas13a combine special Other RNA (designed here as RNA fluorescence reports system) are cut immediately after property target RNA, with isothermal amplification technique Recombinase polymerase amplification (RPA) are combined, and this detection method is called SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing).The above method is related to RNA's Detection, if DNA detection need to be only directed to, in view of RNA unstability, may add operation difficulty.
2015, Zhang Feng et al. was found that new CRISPR GAP-associated protein GAP restriction endonucleases Cas12a (being referred to as Cpf1 before), it Specific DNA endonuclease with as conventional Cas9 albumen being RNA guiding, but the Cas9 compared with, Cas12a have it again Own characteristic, for example the i.e. bootable specific cutting double-stranded DNAs of crRNA are only needed, and produce cohesive end etc..
The content of the invention
It is an object of the invention to provide a kind of detection method of target nucleic acids molecule.
It is a further object of the present invention to provide a kind of purposes of Cas albumen in the detection method of target nucleic acids molecule.
The invention also discloses a kind of kit, including guide RNA, Cas albumen, nucleic acid probe, buffer solution.
A kind of detection method of target nucleic acids molecule, by the reaction system containing target nucleic acids molecule to be checked, guide RNA, Cas albumen, nucleic acid probe, buffer solution, fluoroscopic examination then is carried out to it.
The Cas albumen is Cas12a.
Described Cas12a be preferably FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, It is a kind of in OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a.
Described Cas12a is preferably LbCas12a.
Guide RNA refers to the RNA for guiding Cas protein-specific combination targets DNA.
Target nucleic acids molecule to be checked in the reaction system of target nucleic acids molecule to be checked obtains after amplification.
The detection method can detect pathogenic microorganism, gene mutation or special target DNA.
A kind of purposes of Cas albumen in the detection method of target nucleic acids molecule.
When target DNA, guide RNA and Cas albumen form ternary complex, the compound can be other single in cutting system Ssdna molecule.
Guide RNA refers to the RNA for guiding Cas protein-specific combination targets DNA.
A kind of kit, including guide RNA, Cas albumen, nucleic acid probe.
Also include buffer solution.
Main advantages of the present invention are：
(1) it is quick：In the case of test condition is ready, from taking sample, to take testing result only need it is about 1 small When.
(2) it is inexpensive：There is no special material or enzyme in experiment, and material, less, the Ke Yijin such as reagent being related to The test analysis of row milligram ammonia.
(3) efficiently：The present invention has high sensitivity, can detect the DNA of 10aM concentration.
(4) multichannel：Multi-channel detection form is can be made into, for example reacts and detects in 96 orifice plates or 384 orifice plates.
(5) it is simple：There is no the step of Special complex, if kit is made and sets program, only need to simply add Enter the operation such as sample.
Brief description of the drawings
Fig. 1 shows Cas12a cutting single-chains DNA cis characteristics.
PAM sequences when Fig. 2 shows Cas12a to single stranded DNA cutting independent of cutting double-strand.
Fig. 3 shows Cas12a cutting single-chains DNA trans characteristics.
Fig. 4 shows that the Cas12a of 10 kinds of separate sources of test, these Cas12a have cis and trans vigor.
Fig. 5 is tested by single-site mutant, tests the site that cis to trans vigor may be related.
Fig. 6 shows C2c1 and Cas12a monomers and complex structure.
Fig. 7 shows that different Cas12a utilize specific substrate (dsDNA), is visited using HEX-N12-BHQ1 as fluoroscopic examination Pin, obtained fluorescence values.Control is not added with specific substrate.
Fig. 8 shows HOLMES detections DNA schematic flow sheet.
Fig. 9, which is shown, directly utilizes FnCas12a or LbCas12a, or combines HOLMES methods and carry out sensitivity test.
Figure 10 shows the crRNA combination FnCas12a or LbCas12a of different length to different loci single-site mutant Response.
Figure 11 utilizes the fluorescence probe of FAM marks, and after adding ssDNA from 10 kinds of Cas12a albumen tests, probe is It is no to be cut open.
Figure 12 is shown using gyrB genetic fragments as target sequence using HEX, BHQ1 fluorescence probe marked, with different dense The pure culture Escherichia coli MG1655 of degree is the HOLMES detected values of positive control template, and the inspection of different location environment reclaimed water Measured value.
Figure 13 shows that with the reduction of Escherichia coli MG1655 concentration its fluorescence response value gradually decreases.
Figure 14 shows HOMLES methods detection SNP schematic flow sheet, and 5 SNP site detected values.
Figure 15 shows that HOMLES methods detect the detected value of critical sites in TP53 genes (cancer related gene).
Figure 16 shows that HOMLES methods detect the detected value of 5 SNP sites (gout is related).
Figure 17 shows that HOMLES methods detect the detected value of 1 SNP site (gout is related), and wherein sample is 21 will Hope person's sample.
Embodiment
To make the purpose, technical scheme and advantage of the embodiment of the present invention clearer, below in conjunction with the embodiment of the present invention In accompanying drawing, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is Part of the embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, ordinary skill people The every other embodiment that member is obtained under the premise of creative work is not made, belongs to the scope of protection of the invention.
The present inventor and in-depth study, by the research to Cas12a cleavage characteristics, develops one kind by extensive The technical scheme of target nucleic acids detection.Test result indicates that using being in above-mentioned technical proposal successfully Rapid identification water body It is no to contain the microorganisms such as certain density Escherichia coli, and the Rapid identification of SNP genotype.
Term
" guide RNA " refers to the RNA for guiding Cas protein-specific combination target DNA sequence dnas to term.
Term " crRNA " refers to CRISPR RNA, is short guiding Cas12a to the RNA for being attached to target DNA sequence dna.
Term " CRISPR " refers to cluster, regular intervals short palindrome repetitive sequence (clustered regularly Interspaced short palindromic repeats), the sequence is the immune system of many prokaryotes.
Term " Cas albumen " refers to CRISPR-associated albumen, and it is the GAP-associated protein GAP in CRISPR systems.
Term " Cas12a " (being once called as " Cpf1 ") refers to the restriction endonuclease that crRNA is relied on, and it is V-type in CRISPR genealogical classifications The enzyme of (type V).
Term " PAM " refer to before between region sequence adjacent to motif (protospacer-adjacent motif), be Cas12a Cutting institute is necessary, and FnCas12a PAM is TTN sequences, and LbCas12a PAM is TTTN sequences.
The invention discloses a kind of detection method of target nucleic acids molecule, by the reactant containing target nucleic acids molecule to be checked In system, guide RNA, Cas albumen, nucleic acid probe, buffer solution, fluoroscopic examination then is carried out to it.
The Cas albumen is Cas12a；
Described Cas12a be preferably FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, It is a kind of in OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a；Described Cas12a is preferably LbCas12a。
Guide RNA refers to the RNA for guiding Cas protein-specifics targeting DNA sequence dna.
Target nucleic acids molecule to be checked in the reaction system of target nucleic acids molecule to be checked obtains after amplification.
The detection method can detect pathogenic microorganism, gene mutation or special target DNA.
A kind of purposes of Cas albumen in the detection method of target nucleic acids molecule.
When target DNA, guide RNA and Cas albumen form ternary complex, the compound can be other single in cutting system Ssdna molecule.
Guide RNA refers to the RNA for guiding Cas protein-specifics targeting DNA sequence dna.
A kind of kit, including guide RNA, Cas albumen, nucleic acid probe.
Also include buffer solution.
The invention provides a kind of detection method of the target nucleic acids molecule of quick detection with high specificity.Once target DNA When (single-stranded or double-strand), crRNA and Cas12a albumen form ternary complex, the compound can be other in cutting system Single strand dna.Target DNA (needing the section of DNA sequence detected) is targetted by designing crRNA；Added into detection architecture CrRNA and Cas12a albumen；In the presence of target DNA, Cas12a and crRNA and target DNA form ternary complex, together When the compound exercise its trans cutting activity and cutting belt fluorescence signal mark single stranded DNA (two is connected with hair respectively Light group and quenching group, luminophore can light after being cut off), so as to send fluorescence.Therefore, by detecting fluorescence Learn in system to be detected and whether contain target DNA molecular.Whether the method using the present invention can contain in quick detection sample Specific DNA sequence.By and round pcr combination, the sensitivity of the detection method can be greatly improved.The present invention In accounting probe be preferably fluorescence probe.
HOLMES condition tests：
Cas12a selection：Root it was found that Cas12a have trans cut vigor, once i.e. target DNA, crRNA and , can other single stranded DNAs (bypass DNA) in cutting system when Cas12a albumen forms ternary complex.Set according to this principle Specific DNA detection method is counted.First, bypass DNA being designed to fluorescence probe, its composition is 12nt random sequence, and In 5 ' end mark fluorescent group HEX in 3 ' end mark quenching group BHQ1 (HEX-N12-BHQ1).When containing target in system During DNA fragmentation, the ternary complex of target DNA, crRNA and Cas12a albumen will be formed, now the probe is cut, together When fluorescence (exciting light 535nM, launch light 556nM) can be sent by the detection HEX fluorophors of fluorescence detector.Then, survey 10 kinds of different Cas12a are tried, target sequence is that double-stranded DNA is as shown in Figure 7.As can be seen that target dsDNA with it is each The complex of Cas12a albumen composition can realize that trans cuts vigor.
HOLMES response sensitivities：Then, response sensitivities of the FnCas12a and LbCas12a to target DNA is tested, That is the concentration for the minimum target DNA that can be responded is investigated.As shown in figure 9, when being directly added into test target, 0.1nM is dense Target DNA more than degree is capable of responding to, and is responded during more than concentration 1nM notable.If with reference to round pcr (HOLMES side Method), as shown in figure 8, first pass through PCR amplifications purpose fragment carries out Cas12a cleavage reactions again, now, response sensitivity can With as little as 10aM, as shown in Figure 9.
SNP is tested：Then, whether test HOLMES methods can detect SNP genotype., will using T1 as target sequence The PAM mutation in the site or 1-18 positions target sequence carry out single-site mutant respectively, compare the crRNA of different length to non-prominent It is different to become error of measurement between sequence and mutant nucleotide sequence.As shown in Figure 10, as the crRNA (crRNA- that target-complementary sequence is 24nt When 24nt), the single-site mutant and wild type difference of 8-18 positions are little, and after PAM mutation and 1-7 site mutations, fluorescent value has It is obvious to decline.When truncation crRNA length, when pairing target sequence length is 18nt, mutated site 8-16nt fluorescent value phase It is decreased obviously than 24nt, as 16nt or 17nt, the target sequence fluorescent value decline after mutation is more obvious, and during 15nt, Target sequence and the fluorescent value of mutation are then all very weak.In general, detections of the 16nt and 17nt crRNA to SNP is closed the most It is suitable.
The present invention is not dependent on the journey of PAM sequences to the cutting of Cas12a cutting single-chains DNA, Cas12a to single stranded DNA Sequence cutting mode, referred to as cis are cut；, will and once ternary complex Cas12a/crRNA/target DNA are formed The activity of trans cuttings is shown, i.e., can show any single stranded DNA non-target in cutting system.
Utilize Cas12a characteristic, the method for developing specific nucleic acid detecting molecule, referred to as HOLMES (one HOur Low-cost Multiplex Efficient Simple assay).As the title of the technology, it is characterized in quick (1 hour), low price, multi-path, efficient, easy method of testing.This method can be used for the neck such as quick detection of pathogens, SNP detections Domain.
Material
1.RNA enzyme inhibitors, high-fidelity DNA polymerase KOD FX are purchased from TaKaRa companies；Primer (oligonucleotides) is by upper Hai Shenggong is synthesized；T7 RNA polymerases are purchased from Thermo companies；RNA is purified and concentrated reagent box (RNA Clean＆ ConcentratorTM- 5) Zymo Research are purchased from；SV Gel and PCR Clean-Up System are purchased from Promega companies；Culture medium (e.g., Tryptone, Yeast Extract etc.) is purchased from OXOID companies.
2. culture medium prescription：Liquid LB (1%Tryptone, 0.5%Yeast extract, 1%NaCl), configure solid During LB, it is only necessary to 1% agar is added in liquid LB.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.Involved experiment material unless otherwise specified can be from commercially available channel in the present invention Obtain.
The target (probe FAM marks) of the Cas12a Protein Detections single stranded DNA (ssDNA) of embodiment 1
Target sequence is used as from single stranded DNA (target-T1-R), tests the response that different Cas12a albumen detect to it Value.
1st, prepared by crRNA：First, by using T7-crRNA-F and the oligonucleotides T7-T1-24-R of synthesis, such as the institute of table 5 Show, annealed to prepare transcription templates.Specifically, by the oligonucleotides (4 μM) of pairing in 1 × PCR buffer solutions Annealed in (Transgen Biotech), cumulative volume is 50 μ L, then carries out cycle of annealing：In 95 DEG C of denaturations 5 minutes, Then 20 DEG C are cooled to from 95 DEG C, 1 DEG C is reduced using thermal cycler is per minute.Synthesized using T7 high yield transcript reagents box CrRNA, and react and carried out at 37 DEG C overnight (about 16h).Then using RNA purifying and concentrated reagent box purifying RNA, it is used in combination NanoDrop 2000C (Thermo Fisher Scientific) are quantitative, are diluted to 10 μM of concentration and are saved in -80 DEG C of refrigerators In.
2nd, Cas12a reacts：In 20 μ L reaction systems, the crRNA (0.5 μM), Cas12a (0.25 purified is added in step 1 μM), target single stranded DNA (target-T1-R) (0.01 μM), nucleic acid probe (N25-5 ' FAM) (0.01 μM), buffer solution NEB The μ L RNase inhibitors of buffer 3.1,0.5.Control reaction other compositions all add, and are only not added with ssDNA target sequences.At 37 DEG C 15min is reacted, then 98 DEG C of 2min terminating reactions.
3rd, fluoroscopic examination：By urea-acrylamide gel electrophoresis (Urea-PAGE) electrophoresis, then with fluorescence radiation into As instrument detects.As shown in figure 11, different Cas12a are different to target detection effect.Such as HkCas12a, it is not added with target ssDNA It will also result in the cutting of probe.And LbCas12a etc., only when adding target ssDNA, the cutting of probe occurs, is preferable Candidate's Cas12a albumen.
The target of the Cas12a Protein Detections single stranded DNA (ssDNA) of embodiment 2 (probe has HEX, BHQ1 double labelling)
Target sequence is used as from single stranded DNA (target-T1-R), tests the response that different Cas12a albumen detect to it Value.
1st, prepared by crRNA：First, carried out by using T7-crRNA-F and the oligonucleotides T7-T1-24-R (table 5) of synthesis Anneal to prepare transcription templates.Specifically, by the oligonucleotides (4 μM) of pairing in 1 × PCR buffer solutions (Transgen Biotech annealed in), cumulative volume is 50 μ L, then carries out cycle of annealing：In 95 DEG C of denaturations 5 minutes, then from 95 DEG C 20 DEG C are cooled to, 1 DEG C is reduced using thermal cycler is per minute.CrRNA is synthesized using T7 high yield transcript reagents box, and instead It should be carried out at 37 DEG C overnight (about 16h).Then using RNA purifying and concentrated reagent box purifying RNA, and NanoDrop is used 2000C is quantified, and is diluted to 10 μM of concentration and is saved in -80 DEG C of refrigerators.
2nd, Cas12a reacts：In 20 μ L reaction systems, the crRNA (0.5 μM), Cas12a (0.25 purified is added in step 1 μM), target single stranded DNA (target-T1-R) (0.01 μM), fluorescence probe (HEX-N12-BHQ1, i.e. 12nt single stranded DNA, Its 5 ' end is that HEX is marked, and 3 ' ends mark for BHQ1) (0.5 μM), buffer solution is the suppression of the μ L RNases of NEB buffer 3.1,0.5 Preparation.Control reaction other compositions all add, and are only not added with ssDNA target sequences.15min is reacted at 37 DEG C, then 98 DEG C of 2min ends Only react.
3rd, fluoroscopic examination：20 μ L reaction solutions of inactivation are added in 96 orifice plates, then detect (exciting light with ELIASA 535nm, launch light 556nm).As shown in figure 12, different Cas12a are different to target detection effect.Such as HkCas12a, it is not added with Target ssDNA will also result in the cutting of probe.And FnCas12a etc., only when adding target ssDNA, the cutting of probe occurs, It is preferable candidate Cas12a albumen.
The target of the Cas12a Protein Detections double-stranded DNA (dsDNA) of embodiment 3
Target sequence is used as from double-stranded DNA (target-T1), tests the response that different Cas12a albumen detect to it Value.
1st, prepared by crRNA：First, carried out by using T7-crRNA-F and the oligonucleotides T7-T1-24-R (table 5) of synthesis Anneal to prepare transcription templates.Specifically, by the oligonucleotides (4 μM) of pairing in 1 × PCR buffer solutions (Transgen Biotech annealed in), cumulative volume is 50 μ L, then carries out cycle of annealing：In 95 DEG C of denaturations 5 minutes, then from 95 DEG C 20 DEG C are cooled to, 1 DEG C is reduced using thermal cycler is per minute.CrRNA is synthesized using T7 high yield transcript reagents box, and instead It should be carried out at 37 DEG C overnight (about 16h).Then using RNA purifying and concentrated reagent box purifying RNA, and NanoDrop is used 2000C is quantified, and is diluted to 10 μM of concentration and is saved in -80 DEG C of refrigerators.
2nd, Cas12a reacts：In 20 μ L reaction systems, the crRNA (0.5 μM), Cas12a (0.25 purified is added in step 1 μM), target double-stranded DNA (target-T1, by being obtained after primer target-T1-F and primer target-T1-R annealing) (0.01 μM), fluorescence probe (HEX-N12-BHQ1) (0.5 μM), buffer solution is the μ L RNase inhibitors of NEB buffer 3.1,0.5. 15min is reacted at 37 DEG C, then 98 DEG C of 2min terminating reactions.
3rd, fluoroscopic examination：20 μ L reaction solutions of inactivation are added in 96 orifice plates, then detect (exciting light with ELIASA 535nm, launch light 556nm).As shown in fig. 7, different Cas12a are different to target detection effect.And LbCas12a, only adding When entering target ssDNA, the cutting of probe occurs, is preferable candidate Cas12a albumen.
The FnCas12a of embodiment 4 and LbCas12a test various concentrations targets
From target-T1 as target DNA, then gradient dilution is into various concentrations, test FnCas12a with LbCas12a responses to which sensitivity.In order to increase sensitivity, PCR amplification steps are added.
1st, prepared by crRNA：First, carried out by using T7-crRNA-F and the oligonucleotides T7-T1-24-R (table 5) of synthesis Anneal to prepare transcription templates.Specifically, by the oligonucleotides (4 μM) of pairing in 1 × PCR buffer solutions (Transgen Biotech annealed in), cumulative volume is 50 μ L, then carries out cycle of annealing：In 95 DEG C of denaturations 5 minutes, then from 95 DEG C 20 DEG C are cooled to, 1 DEG C is reduced using thermal cycler is per minute.CrRNA is synthesized using T7 high yield transcript reagents box, and instead It should be carried out at 37 DEG C overnight (about 16h).Then using RNA purifying and concentrated reagent box purifying RNA, and NanoDrop is used 2000C is quantified, and is diluted to 10 μM of concentration and is saved in -80 DEG C of refrigerators.
2nd, PCR expands (optional)：Using the plasmid (pUC18-T1) of the target containing target-T1 as template, gradient dilution, enter Performing PCR reacts.Each reaction system cumulative volume is 20 μ L, and M13F-47, M13R-48 with 0.25 μM are primer (table 4), PCR High-fidelity enzyme KOD FX (TaKaRa) are used in reaction.PCR response procedures are 95 DEG C of 2min, then start 35 circulations, 98 DEG C of 10s, 60 DEG C of 15s, 68 DEG C of 10s.After the completion of PCR, Cas12a reactions are directly used in.
3rd, Cas12a reacts：In 20 μ L reaction systems, add the crRNA (0.5 μM) that purifies in step 1, FnCas12a or LbCas12a (0.25 μM), the μ L of PCR products 1 (or being directly diluted to the target DNA of various concentrations), fluorescence probe (HEX-N12- BHQ1) (0.5 μM), buffer solution are the μ L RNase inhibitors of NEB buffer 3.1,0.5.React 15min at 37 DEG C, then 98 DEG C 2min terminating reactions.
4th, fluoroscopic examination：20 μ L reaction solutions of inactivation are added in 96 orifice plates, then detect (exciting light with ELIASA 535nm, launch light 556nm).As shown in figure 9, when being directly added into test target, target DNA more than 0.1nM concentration can Enough responses, and responded during more than concentration 1nM notable.If with reference to round pcr, that is, first pass through PCR amplifications purpose fragment and enter again Row Cas12a cleavage reactions, now, response sensitivity can be with as little as 10aM.
The FnCas12a of embodiment 5 and LbCas12a measuring unit point mutation targets
From target-T1 as target, Qi PAM areas and 1-18 positions are subjected to single-site mutant respectively, test is several The crRNA of different length, to the response after wild type and single-site mutant.
1st, prepared by crRNA：First, by using T7-crRNA-F and oligonucleotides T7-T1-24-R, T7-T1-15- of synthesis R, T7-T1-16-R, T7-T1-17-R, T7-T1-18-R (table 5) are annealed to prepare transcription templates respectively.Specifically, will The oligonucleotides (4 μM) of pairing is annealed in 1 × PCR buffer solutions (Transgen Biotech), and cumulative volume is 50 μ L, then Carry out cycle of annealing：In 95 DEG C of denaturations 5 minutes, 20 DEG C then are cooled to from 95 DEG C, uses thermal cycler reduction per minute 1℃.CrRNA is synthesized using T7 high yield transcript reagents box, and reacts and is carried out at 37 DEG C overnight (about 16h).Then use RNA purify with concentrated reagent box purifying RNA, and quantified with NanoDrop 2000C, be diluted to 10 μM of concentration and be saved in -80 In DEG C refrigerator.
2nd, PCR is expanded：Template is used as using the plasmid (pUC18-T1) of the target containing target-T1.Each reaction system is overall Product is 20 μ L, and with 0.25 μM of primer M13R-48 and Target-T1-F each mutant primer (table 4), PCR reactions are protected with high True enzyme KOD FX (TaKaRa).PCR response procedures are 95 DEG C of 2 min, then start 35 circulations 98 DEG C of 10s, 60 DEG C of 15s, 68 ℃10s.After the completion of PCR, Cas12a reactions are directly used in.
3rd, Cas12a reacts：In 20 μ L reaction systems, add the crRNA (0.5 μM) that purifies in step 1, FnCas12a or LbCas12a (0.25 μM), the μ L of PCR products 1, fluorescence probe (HEX-N12-BHQ1) (0.5 μM), buffer solution are NEB buffer 3.1,0.5 μ L RNase inhibitors.15min is reacted at 37 DEG C, then 98 DEG C of 2min terminating reactions.
4th, fluoroscopic examination：20 μ L reaction solutions of inactivation are added in 96 orifice plates, then detect (exciting light with ELIASA 535nm, launch light 556nm).As shown in Figure 10, when target-complementary sequence is 24nt crRNA (crRNA-24nt), 8-18 The single-site mutant and wild type difference of position are little, and after PAM mutation and 1-7 site mutations, fluorescent value significantly decreases. When truncating crRNA length, pairing target sequence length is when be 18nt, mutated site 8-16nt fluorescent value compare 24nt have it is bright It is aobvious to decline, as 16nt or 17nt, the target sequence fluorescent value after mutation decline it is more obvious, and during 15nt, target sequence and The fluorescent value of mutation is then all very weak.
The microorganism testing such as Escherichia coli in the environment water of embodiment 6
It is used as from Escherichia coli gyrB genes and detects target, the microorganism such as Escherichia coli is dense in indirectly testing water body Degree.Using Escherichia coli MG1655 as positive control, the content of microorganism in the water (such as sewage and running water) in determination of the environment.
1st, prepared by crRNA：First, enter by using T7-crRNA-F with the oligonucleotides T7-crRNA-gyrB (table 5) synthesized Row is annealed to prepare transcription templates.Specifically, by the oligonucleotides (4 μM) of pairing in 1 × PCR buffer solutions (Transgen Biotech annealed in), cumulative volume is 50 μ L, then carries out cycle of annealing：In 95 DEG C of denaturations 5 minutes, then from 95 DEG C 20 DEG C are cooled to, 1 DEG C is reduced using thermal cycler is per minute.CrRNA is synthesized using T7 high yield transcript reagents box, and instead It should be carried out at 37 DEG C overnight (about 16h).Then using RNA purifying and concentrated reagent box purifying RNA, and NanoDrop is used 2000C is quantified, and is diluted to 10 μM of concentration and is saved in -80 DEG C of refrigerators.
2nd, PCR is expanded：Positive control sample is that Escherichia coli MG1655 is cultivated to OD600When about 0.5, it is with 10 times respectively Template is used as after gradient dilution, sample is ambient water (including muddy water in running water and environment).Each reaction system cumulative volume For 20 μ L, with 0.25 μM of primer gyrB-F and gyrB-R (table 4), high-fidelity enzyme KOD FX (TaKaRa) are used in PCR reactions.PCR Response procedures are 95 DEG C of 2min, then start 35 circulations 98 DEG C of 10s, 60 DEG C of 15s, 68 DEG C of 10s.After the completion of PCR, directly use Reacted in Cas12a.
3rd, Cas12a reacts：In 20 μ L reaction systems, the crRNA (0.5 μM), LbCas12a purified is added in step 1 (0.25 μM), the μ L of PCR primer 1, fluorescence probe (HEX-N12-BHQ1) (0.5 μM), buffer solution are NEB buffer 3.1,0.5 μ L RNase inhibitors.15min is reacted at 37 DEG C, then 98 DEG C of 2min terminating reactions.
4th, fluoroscopic examination：20 μ L reaction solutions of inactivation are added in 96 orifice plates, then detect (exciting light with ELIASA 535nm, launch light 556nm).As shown in Figure 13, as the reduction of Escherichia coli MG1655 concentration, its fluorescence response value are gradual Reduce.Wherein, sample 2,4,5,6 significantly detected microorganism.
The people SNP of embodiment 7 is tested
SNP tests 5 sites for having selected people SNP, respectively rs5028, rs1467558, rs2952768, Rs4363657, rs601338, test the feasibility of HOLMES methods.
1st, prepared by crRNA：First, annealed by using T7-crRNA-F with the oligonucleotides (table 5) synthesized to prepare Transcription templates.Specifically, the oligonucleotides (4 μM) of pairing is annealed in 1 × PCR buffer solutions (Transgen Biotech), Cumulative volume is 50 μ L, then carries out cycle of annealing：In 95 DEG C of denaturations 5 minutes, 20 DEG C are then cooled to from 95 DEG C, is used Thermal cycler is per minute to reduce by 1 DEG C.CrRNA is synthesized using T7 high yield transcript reagents box, and reacts and was carried out at 37 DEG C Night (about 16h).Use RNA Clean＆ConcentratorTM- 5 (Zymo Research) purifying RNAs, and use NanoDrop 2000C is quantified, and is diluted to 10 μM of concentration and is saved in -80 DEG C of refrigerators.
2nd, PCR is expanded：Reaction system cumulative volume is 20 μ L, with 0.25 μM of primer (table 4), 1ng human genome (293T) or oral epithelium mucous membrane is directly scraped as template, PCR, which reacts, uses high-fidelity enzyme KOD FX (TaKarRa).PCR is anti- It is 95 DEG C of 2min to answer program, then starts 35 circulations 98 DEG C of 10 s, 60 DEG C of 15s, 68 DEG C of 10s.After the completion of PCR, it is directly used in Cas12a reacts.(wherein primer 1-rs5028-F-T, 2-rs1467558-F-T, 3-rs2952768-R-C are introduced directly into SNP Corresponding variants)
3rd, Cas12a reacts：In 20 μ L reaction systems, corresponding crRNA (1 μM), LbCas12a (0.5 μM), PCR are added The μ L of product 1, fluorescence probe (HEX-N12-BHQ1) (0.5 μM).37 DEG C are reacted 15min, then 98 DEG C of 2min terminating reactions.
4th, fluoroscopic examination：20 μ L reaction solutions of inactivation are added in 96 orifice plates, then detect (exciting light with ELIASA 535nm, launch light 556nm).Such as Figure 14, only when crRNA corresponds to corresponding target sequence, just there is higher fluorescence response Value, if the mutation in a site, its response will substantially reduce.Size is obtained by fluorescent value and can determine whether corresponding SNP Genotype, these results have also obtained the confirmation of sequencing result.
The cancer related gene of embodiment 8 is tested
TP53 genes have been selected as test cdna, wherein in people's T24 cells, TP53 genes have a nonsense mutation, The gene is caused to inactivate.The normal cell of the locus gene (293T), personal genetic test, and mutation are tested respectively Cell T24.
1st, prepared by crRNA：First, by using T7-crRNA-F and the oligonucleotides T7-crRNA-34-TP53- of synthesis T24-C-16nt and T7-crRNA-34-TP53-T24-G-16nt (table 5) is annealed to prepare transcription templates.Specifically, will The oligonucleotides (4 μM) of pairing is annealed in 1 × PCR buffer solutions (Transgen Biotech), and cumulative volume is 50 μ L, then Carry out cycle of annealing：In 95 DEG C of denaturations 5 minutes, 20 DEG C then are cooled to from 95 DEG C, uses thermal cycler reduction per minute 1℃.CrRNA is synthesized using T7 high yield transcript reagents box, and reacts and is carried out at 37 DEG C overnight (about 16h).Use RNA Clean&ConcentratorTM- 5 (Zymo Research) purifying RNAs, and quantified with NanoDrop 2000C, it is diluted to 10 μM Concentration is simultaneously saved in -80 DEG C of refrigerators.
2nd, PCR is expanded：Reaction system cumulative volume is 20 μ L, with 0.25 μM of primer 34-TP53-T24-F, 34-TP53- T24-R (table 4), 1ng human genome (293T, T24) directly scrape oral epithelium mucous membrane as template, PCR reactions height Fidelity enzyme KOD FX (TaKaRa).PCR response procedures are 95 DEG C of 2 min, then start 35 circulations 98 DEG C of 10s, 60 DEG C of 15s, 68℃10s.After the completion of PCR, Cas12a reactions are directly used in.
3rd, Cas12a reacts：In 20 μ L reaction systems, corresponding crRNA (1 μM), LbCas12a (0.5 μM), PCR are added The μ L of product 1, fluorescence probe (HEX-N12-BHQ1) (0.5 μM).37 DEG C are reacted 15min, then 98 DEG C of 2min terminating reactions.
4th, fluoroscopic examination：20 μ L reaction solutions of inactivation are added in 96 orifice plates, then detect (exciting light with ELIASA 535nM, launch light 556nM).Such as Figure 15, when the numerical value that the normal TP53 genes in the site are the crRNA-C that template detection arrives Apparently higher than crRNA-G, and the crRNA-G for the cell T24 being mutated is then significantly raised.
The people SNP of embodiment 9 tests (gout related gene)
SNP tests 5 sites for having selected people SNP, and these are related to the risk of gout, respectively rs1014290, Rs6449213, rs737267, rs1260326, rs642803, test HOLMES methods.
1st, prepared by crRNA：First, annealed by using T7-crRNA-F with the oligonucleotides (table 5) synthesized to prepare Transcription templates.Specifically, the oligonucleotides (4 μM) of pairing is annealed in 1 × PCR buffer solutions (Transgen Biotech), Cumulative volume is 50 μ L, then carries out cycle of annealing：In 95 DEG C of denaturations 5 minutes, 20 DEG C are then cooled to from 95 DEG C, is used Thermal cycler is per minute to reduce by 1 DEG C.CrRNA is synthesized using T7 high yield transcript reagents box, and reacts and was carried out at 37 DEG C Night (about 16h).Using RNA Clean＆ConcentratorTM-5 (Zymo Research) purifying RNA, and use NanoDrop 2000C is quantified, and is diluted to 10 μM of concentration and is saved in -80 DEG C of refrigerators.
2nd, PCR is expanded：Reaction system cumulative volume is 20 μ L, with 0.25 μM of primer (table 4), 1ng human genome (293T) or oral epithelium mucous membrane is directly scraped as template, PCR, which reacts, uses high-fidelity enzyme KOD FX (TaKaRa).PCR reacts Program is 95 DEG C of 2min, then starts 35 circulations 98 DEG C of 10s, 60 DEG C of 15s, 68 DEG C of 10s.After the completion of PCR, it is directly used in Cas12a reacts.(wherein primer 1-rs5028-F-T, 2-rs1467558-F-T, 3-rs2952768-R-C are introduced directly into SNP Corresponding variants)
3rd, Cas12a reacts：In 20 μ L reaction systems, corresponding crRNA (1 μM), LbCas12a (0.5 μM), PCR are added The μ L of product 1, fluorescence probe (HEX-N12-BHQ1) (0.5 μM).37 DEG C are reacted 15min, then 98 DEG C of 2min terminating reactions.
4th, fluoroscopic examination：20 μ L reaction solutions of inactivation are added in 96 orifice plates, then detect (exciting light with ELIASA 535nm, launch light 556nm).Such as Figure 16, only when crRNA corresponds to corresponding target sequence, just there is higher fluorescence response Value, if the mutation in a site, its response will substantially reduce.Size is obtained by fluorescent value and can determine whether corresponding SNP Genotype, these results have also obtained the confirmation of sequencing result.
The kit SNP of embodiment 10 test volunteer's clinical samples (gout related gene)
Premixed liquid is added in 96 orifice plates, is prepared into kit, then adds the genomic DNA of 21 volunteers, is surveyed Rs1014290 sites are tried, the site is related to the risk of gout.
1st, prepared by kit：First, annealed by using T7-crRNA-F with the oligonucleotides (table 5) synthesized to prepare Transcription templates.Specifically, the oligonucleotides (4 μM) of pairing is annealed in 1 × PCR buffer solutions (Transgen Biotech), Cumulative volume is 50 μ L, then carries out cycle of annealing：In 95 DEG C of denaturations 5 minutes, 20 DEG C are then cooled to from 95 DEG C, is used Thermal cycler is per minute to reduce by 1 DEG C.CrRNA is synthesized using T7 high yield transcript reagents box, and reacts and was carried out at 37 DEG C Night (about 16h).Using RNA Clean＆ConcentratorTM-5 (Zymo Research) purifying RNA, and use NanoDrop 2000C is quantified, and is diluted to 10 μM of concentration.
2nd, 96 orifice plate PCR are premixed：In 19 μ L systems, reagent needed for PCR reactions, primer 41-rs1014290-F are added With 41-rs1014290-R.
3rd, the orifice plate of fluoroscopic examination 96 premixes：In 19 μ L systems, crRNA (1 μM), LbCas12a (0.5 μM), fluorescence are added Probe (HEX-N12-BHQ1) (0.5 μM), is added in 96 orifice plates.
4th, PCR is expanded：Volunteer's genomic DNA is added in 96 orifice plates of PCR premixs, then enters performing PCR reaction.PCR Response procedures are 95 DEG C of 2min, then start 35 circulations 98 DEG C of 10s, 60 DEG C of 15s, 68 DEG C of 10s.
5th, Cas12a reacts：1 μ L PCR reaction solutions are taken, are added in the orifice plate of fluoroscopic examination 96 premixed, 37 DEG C of reactions 15min, then 98 DEG C of 2min terminating reactions.
6th, fluoroscopic examination：Detected with ELIASA and (exciting light 535nm, launched light 556nm).Such as Figure 17, due to genotype A's Crowd has higher gout risk, therefore outside 5,7,9 st volunteers, other people need to be important to note that the risk of gout.
Cas12a cuts ssDNA cis cutting characteristics：
First, Cas12a ssDNA Cutting features, the crRNA (tables of several short ssDNA (DNMT1-3) of targeting are devised 1), it is marked in 3' ends with 5 (6)-Fluoresceincarboxylic acid (FAM).After FnCas12a cuttings, pass through denaturing urea polypropylene Acrylamide gel electrophoresis (urea PAGE) analytical reactions product.It was found that the ssDNA cuttings for passing through Cas12a are sequencing, that is, cut It is near the 22nd base matched from first 3'- base with crRNA homing sequences (the 21st to the 23rd), such as to cut site Shown in Figure 1A and 1C.Cas12a dsDNA needs PAM sequences, and ssDNA cuttings do not need PAM sequences (Figure 1A, 1B and figure 2), this is similar to the ssDNA cuttings of Cas9 mediations.However, the ssDNA cleavage activities of Cas12a mediations are depended in crRNA Loop-stem structure, as shown in Figure 1A, and Cas9 is still shown to only having the ssDNA's of 20-nt complementary RNA sequences still to have weak cut Cut activity.CrRNA loop-stem structure is important for the structure for stablizing Cas12a, and it is crRNA ring structures to Cas12a's The reason for necessity of ssDNA cuttings.Whether further test Cas12a ssDNA cleavage sites can be drawn by shorter Sequence crRNA is led, so that cutting is outside recognition site.When homing sequence length is 16nt, 18nt and 20nt, institute There are these crRNA to result in cutting by Cpf1 at the 22nd base, as shown in Figure 1B and 1D, it is meant that cleavage Point is 4nt, 2nt or the 0nt outside recognition site.Then, cutting efficiencies of the Cas12a to different substrates is tested, is used respectively DsDNA and ssDNA substrates, as shown in fig. 1F.Situation is cut similar to Cas9, slower, such as figure is cut in ssDNA cuttings than dsDNA Shown in 1E and 1G.The mechanism that these results show Cas12a ssDNA identifications and cutting likely differs from dsDNA, is a kind of effect The relatively low identification cutting mode independent of PAM of rate；PAM sequences play for identifications and/or cutting of the Cas12a to target to be added The effect of speed.
Cas12a cuts ssDNA trans cutting characteristics：
When ssDNA targets are in 3' end marks, Cas12a is cut near the 22nd base, as shown in Figure 1.However, work as Band is not observed at 5' ends in mark at the size of prediction, but produce it is short (<6nt) the product of FAM marks, such as Shown in Fig. 3 B.By detailed experiment, once ternary complex Cas12a/crRNA/target ssDNA are formed, 5'- ends The target ssDNA (DNMT1-3) (table 1) of mark is cut and produces the product of short FAM marks, as shown in Figure 3 C.In addition, three First complex is also cut does not have the ssDNAs of complementary sequence with crRNA in any other reaction system, such as Fig. 3 C and Fig. 3 D institutes Show.This cutting phenomenon is cut for trans, is different from the cis cuttings of programmable.When target ssDNA is in 3'- end marks When, trans cuttings are also observed, but many cis cleaved products are left, as shown in Figure 3 B, this is probably by Cas12a/ The compound of crRNA/target ssDNA formation, the target ssDNA 3'- ends of mark are protected from exposure to core Phytase activity site, these cutting process can be shown with Fig. 3 A.
Except the FnCas12a tested above, 9 kinds of Cas12a (table 2 and figure from other source of species are also tested for 4A).In addition to Lb4Cas12a, all Cas12a have preferable endonuclease activity to DNA, such as Fig. 4 B Shown and all Cas12a ternary complexs are all shown to single-stranded cis and trans cleavage activities, such as Fig. 4 C and 4D It is shown.This explanation Cas12a cis and trans vigor to single stranded DNA is universal phenomenon.
Cas12a cuts ssDNA cis and trans critical sites and mechanism
In order to determine in Cas12a about the critical amino acid residues to single-stranded cis and trans activity, Cas12a has been mutated Several Candidate Residues carry out vigor test.First, purify and test three of FnCas12a (H843A, K852A and K869A) Single amino acid mutations body, its residue are related to RNase activity.Shown for ssDNA trans activity research results, wild type FnCas12a and three mutant does not discover a marked discrepancy on cis and trans cutting vigor, as seen in figs. 5 a and 5 c.
Then, when the endonuclease activity site mutation in FnCas12a, i.e. RuvC domains (D917A, E1006A Or D1255A) be affected with Nuc domains (R1218A) site, these mutation Cas12a cis and trans activity, As figs. 5 b and 5 d show.These results show that the critical sites of dsDNA cuttings and cis and trans activity are closely related.
Structural research to C2c1 recently (including with the target DNA of extension or the non-target DNA complex of extension) display two Chain is respectively positioned in RuvC pockets, as shown in Figure 6 A and 6B.By comparing C2c1 and Cas12a endonuclease catalysed residue, These site most probables are in C2c1 and Cas12a the cutting effect similar with being played in function.External Single amino acid mutations are real The result tested shows, is consistent with above-mentioned hypothesis, in other words Cas12a is catalyzed mouth likely via only passing through a RuvC Bag two chains of cutting.
The trans activity of Cas12a complexs：In C2c1 complexs and extra ssDNA structure, sequence-independent manner SsDNA also is located at the surface of catalytic pocket, as shown in Figure 6 C, the trans ssDNA substrates being similarly in Cas12a.With reference to Single amino acids mutating experiment, propose the single RuvC of target DNA, non-target DNA and trans ssDNA all in Cas12a Pocket is cut, as shown in Fig. 6 D, 6E and 6F.Ternary Cas12a complexs have that trans is active, and monomer or binary complex There is no the reason for trans activity to be explained by comparing the structure of monomer, binary and ternary complex.Monomer Cas12a Structure is unordered, and binary complex Cas12a/crRNA is triangular structure, as shown in Figure 6 G, and ternary complex Cas12a/crRNA/target DNA are changed into bi-lobate structure, so as to expose catalytic pocket, as shown in figure 6h.
The foundation of nuclei acid probe method
Utilize Cas12a characteristic, the method for developing specific nucleic acid detecting molecule, referred to as HOLMES (one HOur Low-cost Multiplex Efficient Simple assay).As the title of the technology, it is characterized in quick (1 hour), low price, multi-path, efficient, easy method of testing.
In whole reaction system, two big steps can be divided into, one is amplification to template nucleic acid, and another is The specific nucleic acid detection of Cas12a albumen.Here, the method that PCR has been used in the amplification for nucleic acid, but it is in fact, any Amplification method can all combine the detection of nucleic acids of second step, such as isothermal amplification method RPA etc..Initial nucleic acid is not limited to double Chain DNA or single stranded DNA；Even RNA, can also be detected by being realized after reverse transcription, therefore this method is applied to Polytype nucleic acid molecules.For the detection of nucleic acids stage, wherein 3 components are the keys of experiment, respectively Cas12a, CrRNA and nucleic acid probe.Except the 10 kinds of Cas12a (this 10 kinds of albumen are randomly selected) mentioned in embodiment, other Cas12a albumen is equally applicable to this method.In addition, other types of Cas albumen (such as C2c1 albumen) is also applied for the present invention The category of protection：Shown according to experimental result, Alicyclobacillus acidoterrestris C2c1 also have with Trans vigor similar Cas12a, itself and crRNA/ targets DNA compound can also cut bypass single stranded DNA.
For the crRNA as guiding function, after the transformation such as manually modified, can more stablize in system.In core In the selection of acid probe, the present invention has selected the short ssDNA that HEX and BHQ1 is marked, other detectable any mark mode reasons By it is upper be all it is applicable, as long as the nucleic acid probe be cut after produce detectable difference.Or nucleic acid probe can also be set Count into can combined with compound after fluoresce, so as to detect whether that the probe is cut off.
In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can be to this hair Bright to make various changes or modifications, these equivalent form of values equally fall within the application appended claims limited range.
The related cutting substrate of the Cas12a characteristic tests of table 1
The Cas12a albumen and the title of C2c1 albumen that are related in this patent of table 2. with No. GI
The plasmid used in this patent of table 3
The primer used in the test of the HOLMES methods of table 4
Table 5 is used for the template sequence for transcribing crRNA
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.
SEQUENCE LISTING
<110>Reveal port bio tech ltd in Shanghai
<120>A kind of purposes of Cas albumen and the detection method and kit of target nucleic acids molecule
<130> 2017
<160> 111
<170> PatentIn version 3.3
<210> 1
<211> 50
<212> DNA
<213>Artificial sequence
<400> 1
aatgtttcct gatggtccat gtctgttact cgcctgtcaa gtggcgtgac 50
<210> 2
<211> 50
<212> DNA
<213>Artificial sequence
<400> 2
gtcacgccac ttgacaggcg agtaacagac atggaccatc aggaaacatt 50
<210> 3
<211> 66
<212> DNA
<213>Artificial sequence
<400> 3
tttctgtttg ttatcgcaac tttctactga attcaagctt tactctagaa agaggagaaa 60
ggatcc 66
<210> 4
<211> 66
<212> DNA
<213>Artificial sequence
<400> 4
ggatcctttc tcctctttct agagtaaagc ttgaattcag tagaaagttg cgataacaaa 60
cagaaa 66
<210> 5
<211> 11
<212> DNA
<213>Artificial sequence
<400> 5
acaaacagaa a 11
<210> 6
<211> 16
<212> DNA
<213>Artificial sequence
<400> 6
cgataacaaa cagaaa 16
<210> 7
<211> 22
<212> DNA
<213>Artificial sequence
<400> 7
aagttgcgat aacaaacaga aa 22
<210> 8
<211> 28
<212> DNA
<213>Artificial sequence
<400> 8
agtagaaagt tgcgataaca aacagaaa 28
<210> 9
<211> 34
<212> DNA
<213>Artificial sequence
<400> 9
gaattcagta gaaagttgcg ataacaaaca gaaa 34
<210> 10
<211> 24
<212> DNA
<213>Artificial sequence
<400> 10
gaattcagta gaaagttgcg ataa 24
<210> 11
<211> 18
<212> DNA
<213>Artificial sequence
<400> 11
agtagaaagt tgcgataa 18
<210> 12
<211> 12
<212> DNA
<213>Artificial sequence
<400> 12
aagttgcgat aa 12
<210> 13
<211> 28
<212> DNA
<213>Artificial sequence
<400> 13
cacaattcca cacaacatac gagccgga 28
<210> 14
<211> 27
<212> DNA
<213>Artificial sequence
<400> 14
tgtagccgta gttaggccac cacttca 27
<210> 15
<211> 31
<212> DNA
<213>Artificial sequence
<400> 15
agttttgtta tcgcaacttt ctactgaatt c 31
<210> 16
<211> 31
<212> DNA
<213>Artificial sequence
<400> 16
agttttgata tcgcaacttt ctactgaatt c 31
<210> 17
<211> 31
<212> DNA
<213>Artificial sequence
<400> 17
agttttgtaa tcgcaacttt ctactgaatt c 31
<210> 18
<211> 31
<212> DNA
<213>Artificial sequence
<400> 18
agttttgttt tcgcaacttt ctactgaatt c 31
<210> 19
<211> 31
<212> DNA
<213>Artificial sequence
<400> 19
agttttgtta acgcaacttt ctactgaatt c 31
<210> 20
<211> 31
<212> DNA
<213>Artificial sequence
<400> 20
agttttgtta tggcaacttt ctactgaatt c 31
<210> 21
<211> 31
<212> DNA
<213>Artificial sequence
<400> 21
agttttgtta tcccaacttt ctactgaatt c 31
<210> 22
<211> 31
<212> DNA
<213>Artificial sequence
<400> 22
agttttgtta tcggaacttt ctactgaatt c 31
<210> 23
<211> 31
<212> DNA
<213>Artificial sequence
<400> 23
agttttgtta tcgctacttt ctactgaatt c 31
<210> 24
<211> 31
<212> DNA
<213>Artificial sequence
<400> 24
agttttgtta tcgcatcttt ctactgaatt c 31
<210> 25
<211> 31
<212> DNA
<213>Artificial sequence
<400> 25
agttttgtta tcgcaagttt ctactgaatt c 31
<210> 26
<211> 29
<212> DNA
<213>Artificial sequence
<400> 26
aaaagttatc gcaactttct actgaattc 29
<210> 27
<211> 39
<212> DNA
<213>Artificial sequence
<400> 27
agttttgtta tcgcaacatt ctactgaatt cggtcatag 39
<210> 28
<211> 39
<212> DNA
<213>Artificial sequence
<400> 28
agttttgtta tcgcaactat ctactgaatt cggtcatag 39
<210> 29
<211> 39
<212> DNA
<213>Artificial sequence
<400> 29
agttttgtta tcgcaactta ctactgaatt cggtcatag 39
<210> 30
<211> 39
<212> DNA
<213>Artificial sequence
<400> 30
agttttgtta tcgcaacttt gtactgaatt cggtcatag 39
<210> 31
<211> 39
<212> DNA
<213>Artificial sequence
<400> 31
agttttgtta tcgcaacttt caactgaatt cggtcatag 39
<210> 32
<211> 39
<212> DNA
<213>Artificial sequence
<400> 32
agttttgtta tcgcaacttt cttctgaatt cggtcatag 39
<210> 33
<211> 39
<212> DNA
<213>Artificial sequence
<400> 33
agttttgtta tcgcaacttt ctagtgaatt cggtcatag 39
<210> 34
<211> 39
<212> DNA
<213>Artificial sequence
<400> 34
agttttgtta tcgcaacttt ctacagaatt cggtcatag 39
<210> 35
<211> 31
<212> DNA
<213>Artificial sequence
<400> 35
agtttagtta tcgcaacttt ctactgaatt c 31
<210> 36
<211> 31
<212> DNA
<213>Artificial sequence
<400> 36
agttatgtta tcgcaacttt ctactgaatt c 31
<210> 37
<211> 31
<212> DNA
<213>Artificial sequence
<400> 37
agtattgtta tcgcaacttt ctactgaatt c 31
<210> 38
<211> 28
<212> DNA
<213>Artificial sequence
<400> 38
agttgtcgtt cctcaactcc ggcgtttc 28
<210> 39
<211> 28
<212> DNA
<213>Artificial sequence
<400> 39
tcgacgccaa taccgtcttt ttcagtgg 28
<210> 40
<211> 26
<212> DNA
<213>Artificial sequence
<400> 40
ctgcctttgc ttctaccttt gcctgt 26
<210> 41
<211> 25
<212> DNA
<213>Artificial sequence
<400> 41
ttgcttctac ctttgcctgt tctgg 25
<210> 42
<211> 25
<212> DNA
<213>Artificial sequence
<400> 42
ttttctggct ggggatggcc gatgg 25
<210> 43
<211> 50
<212> DNA
<213>Artificial sequence
<400> 43
agcaataaca ctaatattga ttccttcaga tatggactcc tttcatagta 50
<210> 44
<211> 39
<212> DNA
<213>Artificial sequence
<400> 44
ttgattcctt cagatatgga ctcctttcat agtataacg 39
<210> 45
<211> 37
<212> DNA
<213>Artificial sequence
<400> 45
tgagcatcgt tattcttacg cgttgtcatt gaaagag 37
<210> 46
<211> 25
<212> DNA
<213>Artificial sequence
<400> 46
agcctgggca acgagtgaaa ctctg 25
<210> 47
<211> 27
<212> DNA
<213>Artificial sequence
<400> 47
acaggaggga caaaggccta agtgtcc 27
<210> 48
<211> 38
<212> DNA
<213>Artificial sequence
<400> 48
catcatagga ttgggaaaag gacatttcag tcattcag 38
<210> 49
<211> 48
<212> DNA
<213>Artificial sequence
<400> 49
agagtccttc tttctcaatt tttcagaata atttagtact ttgggtac 48
<210> 50
<211> 40
<212> DNA
<213>Artificial sequence
<400> 50
cagtactgaa aaaacctgcc tatcaataaa agccctagac 40
<210> 51
<211> 24
<212> DNA
<213>Artificial sequence
<400> 51
gcttcaccgg ctacctttgc tcct 24
<210> 52
<211> 21
<212> DNA
<213>Artificial sequence
<400> 52
ttcacctgca ggccccgcag g 21
<210> 53
<211> 39
<212> DNA
<213>Artificial sequence
<400> 53
cctgactttc aactctgtct ccttcctctt tttacagta 39
<210> 54
<211> 28
<212> DNA
<213>Artificial sequence
<400> 54
tgctgtgact gcttgtagat ggccatgg 28
<210> 55
<211> 36
<212> DNA
<213>Artificial sequence
<400> 55
agtttccaga cctcagtgca caagatactt ttctac 36
<210> 56
<211> 36
<212> DNA
<213>Artificial sequence
<400> 56
acctcagtgc acaagatact tttctacgtc atccac 36
<210> 57
<211> 33
<212> DNA
<213>Artificial sequence
<400> 57
agctccagtg gatggaagat ctttgagatc cag 33
<210> 58
<211> 40
<212> DNA
<213>Artificial sequence
<400> 58
agtcaaagag attcatgcct gggactttaa tcacatttat 40
<210> 59
<211> 33
<212> DNA
<213>Artificial sequence
<400> 59
atgcctggga ctttaatcac atttatcgga agg 33
<210> 60
<211> 31
<212> DNA
<213>Artificial sequence
<400> 60
caaatctgtc tccacctctc agctcacctt g 31
<210> 61
<211> 34
<212> DNA
<213>Artificial sequence
<400> 61
ttcttgaacc caaactcacc tggcatttaa actg 34
<210> 62
<211> 33
<212> DNA
<213>Artificial sequence
<400> 62
aaactcacct ggcatttaaa ctgactctgt aag 33
<210> 63
<211> 33
<212> DNA
<213>Artificial sequence
<400> 63
aaactcacct ggcatttaaa ctgtctctgt aag 33
<210> 64
<211> 27
<212> DNA
<213>Artificial sequence
<400> 64
tgccgaggct gagttcagct actctcc 27
<210> 65
<211> 26
<212> DNA
<213>Artificial sequence
<400> 65
acacagcacc gtgggtcaga ccttgc 26
<210> 66
<211> 26
<212> DNA
<213>Artificial sequence
<400> 66
tgggtcagac tttgccggtg agagtc 26
<210> 67
<211> 26
<212> DNA
<213>Artificial sequence
<400> 67
tgggtcagac tttgctggtg agagtc 26
<210> 68
<211> 27
<212> DNA
<213>Artificial sequence
<400> 68
agcagtggcc atgtgatgct gatgatg 27
<210> 69
<211> 27
<212> DNA
<213>Artificial sequence
<400> 69
ccccggctct gttggctttg agaattg 27
<210> 70
<211> 33
<212> DNA
<213>Artificial sequence
<400> 70
ctctgttggc tttgagaatt gcctgtctgt gtc 33
<210> 71
<211> 33
<212> DNA
<213>Artificial sequence
<400> 71
ctctgttggc tttgagaatt gtctgtctgt gtc 33
<210> 72
<211> 25
<212> DNA
<213>Artificial sequence
<400> 72
accgatacct ggcagccctt ggatg 25
<210> 73
<211> 25
<212> DNA
<213>Artificial sequence
<400> 73
gaaattaata cgactcacta taggg 25
<210> 74
<211> 68
<212> DNA
<213>Artificial sequence
<400> 74
gaattcagta gaaagttgcg ataaatctac aacagtagaa attccctata gtgagtcgta 60
ttaatttc 68
<210> 75
<211> 59
<212> DNA
<213>Artificial sequence
<400> 75
agaaagttgc gataaatcta caacagtaga aattccctat agtgagtcgt attaatttc 59
<210> 76
<211> 60
<212> DNA
<213>Artificial sequence
<400> 76
tagaaagttg cgataaatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60
<210> 77
<211> 61
<212> DNA
<213>Artificial sequence
<400> 77
gtagaaagtt gcgataaatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 78
<211> 62
<212> DNA
<213>Artificial sequence
<400> 78
agtagaaagt tgcgataaat ctacaacagt agaaattccc tatagtgagt cgtattaatt 60
tc 62
<210> 79
<211> 67
<212> DNA
<213>Artificial sequence
<400> 79
gagtaacaga catggaccat cagatctaca acagtagaaa ttccctatag tgagtcgtat 60
taatttc 67
<210> 80
<211> 67
<212> DNA
<213>Artificial sequence
<400> 80
gacatggacc atcaggaaac attatctaca acagtagaaa ttccctatag tgagtcgtat 60
taatttc 67
<210> 81
<211> 67
<212> DNA
<213>Artificial sequence
<400> 81
aggcgagtaa cagacatgga ccaatctaca acagtagaaa ttccctatag tgagtcgtat 60
taatttc 67
<210> 82
<211> 67
<212> DNA
<213>Artificial sequence
<400> 82
tgacaggcga gtaacagaca tggatctaca acagtagaaa ttccctatag tgagtcgtat 60
taatttc 67
<210> 83
<211> 60
<212> DNA
<213>Artificial sequence
<400> 83
agacatggac catcagatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60
<210> 84
<211> 62
<212> DNA
<213>Artificial sequence
<400> 84
acagacatgg accatcagat ctacaacagt agaaattccc tatagtgagt cgtattaatt 60
tc 62
<210> 85
<211> 64
<212> DNA
<213>Artificial sequence
<400> 85
taacagacat ggaccatcag atctacaaca gtagaaattc cctatagtga gtcgtattaa 60
tttc 64
<210> 86
<211> 48
<212> DNA
<213>Artificial sequence
<400> 86
gacatggacc atcaggaaac attccctata gtgagtcgta ttaatttc 48
<210> 87
<211> 48
<212> DNA
<213>Artificial sequence
<400> 87
aggcgagtaa cagacatgga ccaccctata gtgagtcgta ttaatttc 48
<210> 88
<211> 48
<212> DNA
<213>Artificial sequence
<400> 88
tgacaggcga gtaacagaca tggccctata gtgagtcgta ttaatttc 48
<210> 89
<211> 61
<212> DNA
<213>Artificial sequence
<400> 89
cctcttccca gaacaggatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 90
<211> 61
<212> DNA
<213>Artificial sequence
<400> 90
cctcttccca gcacaggatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 91
<211> 61
<212> DNA
<213>Artificial sequence
<400> 91
ctgaagcgtt atactatatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 92
<211> 61
<212> DNA
<213>Artificial sequence
<400> 92
ctgaagcgtt gtactatatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 93
<211> 60
<212> DNA
<213>Artificial sequence
<400> 93
ttttatctga atgattatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60
<210> 94
<211> 60
<212> DNA
<213>Artificial sequence
<400> 94
ttttatctga atgactatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60
<210> 95
<211> 61
<212> DNA
<213>Artificial sequence
<400> 95
aaaaaagagt gagtaccatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 96
<211> 61
<212> DNA
<213>Artificial sequence
<400> 96
aaaaaagagt gggtaccatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 97
<211> 61
<212> DNA
<213>Artificial sequence
<400> 97
ggtagaaggt ccaggagatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 98
<211> 61
<212> DNA
<213>Artificial sequence
<400> 98
ggtagaaggt ctaggagatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 99
<211> 60
<212> DNA
<213>Artificial sequence
<400> 99
gggcagggga gtactgatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60
<210> 100
<211> 60
<212> DNA
<213>Artificial sequence
<400> 100
gggcagggga ctactgatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60
<210> 101
<211> 59
<212> DNA
<213>Artificial sequence
<400> 101
tcagtggatg atgtaatcta caacagtaga aattccctat agtgagtcgt attaatttc 59
<210> 102
<211> 59
<212> DNA
<213>Artificial sequence
<400> 102
tcagtggatg acgtaatcta caacagtaga aattccctat agtgagtcgt attaatttc 59
<210> 103
<211> 61
<212> DNA
<213>Artificial sequence
<400> 103
ggaaattctc cttccgaatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 104
<211> 61
<212> DNA
<213>Artificial sequence
<400> 104
ggaaattctc cttccaaatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 105
<211> 60
<212> DNA
<213>Artificial sequence
<400> 105
tcttacagag tcagttatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60
<210> 106
<211> 60
<212> DNA
<213>Artificial sequence
<400> 106
tcttacagag ccagttatct acaacagtag aaattcccta tagtgagtcg tattaatttc 60
<210> 107
<211> 61
<212> DNA
<213>Artificial sequence
<400> 107
gtcttacaga gacagttatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 108
<211> 59
<212> DNA
<213>Artificial sequence
<400> 108
ctggactctc accagatcta caacagtaga aattccctat agtgagtcgt attaatttc 59
<210> 109
<211> 61
<212> DNA
<213>Artificial sequence
<400> 109
cacagacagg caattctatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 110
<211> 61
<212> DNA
<213>Artificial sequence
<400> 110
cacagacaga caattctatc tacaacagta gaaattccct atagtgagtc gtattaattt 60
c 61
<210> 111
<211> 68
<212> DNA
<213>Artificial sequence
<400> 111
tcgcgcttgt cgcgcagacg aatgatctac aacagtagaa attccctata gtgagtcgta 60
ttaatttc 68

Claims (10)

1. a kind of detection method of target nucleic acids molecule, it is characterised in that add into the system containing target nucleic acids molecule to be checked Enter guide RNA, Cas albumen, nucleic acid probe, buffer solution, then nucleic acid probe is detected.
2. the detection method of target nucleic acids molecule according to claim 1, it is characterised in that the Cas albumen is Cas12a has the Cas albumen similar with Cas12a bypass single stranded DNA cleavage activity；
Described Cas12a be preferably FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, It is a kind of in TsCas12a, BbCas12a, BoCas12a or Lb4Cas12a；Described Cas12a is preferably LbCas12a.
3. the detection method of target nucleic acids molecule according to claim 1, it is characterised in that guide RNA refers to guide Cas Protein-specific combination target DNA RNA.
4. the detection method of target nucleic acids molecule according to claim 1, it is characterised in that the nucleic acid probe is single-stranded DNA；Described single stranded DNA is preferably the single stranded DNA of fluorescence labeling；Described single stranded DNA is preferably to hold mark fluorescent base 5 ' Group HEX and the fluorescence probe after 3 ' end mark quenching group BHQ1；Preferably,
The detection method of the nucleic acid probe is preferably fluorescence detection；Described fluorescence detection be preferably using ELIASA or The method that person's sepectrophotofluorometer is detected.
5. the detection method of target nucleic acids molecule as claimed in any of claims 1 to 4, it is characterised in that to be checked Target nucleic acids molecule to be checked in the reaction system of target nucleic acids molecule is obtained by amplification.
6. the detection method of target nucleic acids molecule according to claim 5, it is characterised in that the detectable disease of the detection method Pathogenic microorganism, gene mutation or special target DNA.
A kind of 7. purposes of Cas albumen in the detection method of target nucleic acids molecule.
8. purposes according to claim 7, it is characterised in that target DNA, guide RNA and Cas albumen form tri compound During body, the compound can other single strand dnas in cutting system；Preferably, guide RNA refers to guide Cas albumen special Property combination target DNA RNA.
9. a kind of detection kit of target nucleic acids molecule, it is characterised in that including guide RNA, Cas albumen, nucleic acid probe.
10. kit according to claim 9, it is characterised in that also including buffer solution.
CN201710573752.0A 2017-07-14 2017-07-14 Application of Cas protein, and detection method and kit of target nucleic acid molecule Active CN107488710B (en)

Priority Applications (23)

Application Number Priority Date Filing Date Title
CN202011092324.4A CN112501254B (en) 2017-07-14 2017-07-14 Application of Cas protein, detection method of target nucleic acid molecule and kit
CN201710573752.0A CN107488710B (en) 2017-07-14 2017-07-14 Application of Cas protein, and detection method and kit of target nucleic acid molecule
MX2020000481A MX2020000481A (en) 2017-07-14 2018-04-12 Application of cas protein, method for detecting target nucleic acid molecule and kit.
CA3069788A CA3069788A1 (en) 2017-07-14 2018-04-12 Use of cas protein, method for detecting target nucleic acid molecule, and kit
BR112020000809-5A BR112020000809A2 (en) 2017-07-14 2018-04-12 use of cas protein, method for detecting nucleic acid molecules, and kit
JP2020523474A JP2020530779A (en) 2017-07-14 2018-04-12 Use of Cas protein, method of detecting target nucleic acid molecule, and kit
SG11202000336RA SG11202000336RA (en) 2017-07-14 2018-04-12 Application of cas protein, method for detecting target nucleic acid molecule and kit
KR1020207003220A KR20200035956A (en) 2017-07-14 2018-04-12 Uses of CAS proteins, methods for detecting target nucleic acid molecules, and kits
AU2018299445A AU2018299445B2 (en) 2017-07-14 2018-04-12 Application of Cas protein, method for detecting target nucleic acid molecule and kit
EA202090290A EA202090290A1 (en) 2017-07-14 2018-04-12 APPLICATION OF PROTEIN Cas, METHOD FOR DETECTING TARGET NUCLEIC ACID MOLECULE AND KIT
PCT/CN2018/082769 WO2019011022A1 (en) 2017-07-14 2018-04-12 Application of cas protein, method for detecting target nucleic acid molecule and kit
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Cited By (70)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108220400A (en) * 2018-01-02 2018-06-29 武汉启丰生物技术有限公司 A kind of quick detection of nucleic acids sizing technique based on CRISPR-Cas10 nucleic acid-protein compounds
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WO2019011022A1 (en) * 2017-07-14 2019-01-17 上海吐露港生物科技有限公司 Application of cas protein, method for detecting target nucleic acid molecule and kit
CN109251963A (en) * 2018-11-12 2019-01-22 复旦大学 The method and kit of mycoplasma contamination in Constant Temperature Detection cell culture fluid
US10253365B1 (en) 2017-11-22 2019-04-09 The Regents Of The University Of California Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs
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WO2020042901A1 (en) * 2018-08-30 2020-03-05 杭州杰毅生物技术有限公司 Crispr-cas-based isothermal method for detecting nucleic acid and kit
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WO2021154866A1 (en) 2020-01-27 2021-08-05 Sherlock Biosciences Improved detection assays
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WO2021155775A1 (en) * 2020-02-03 2021-08-12 苏州克睿基因生物科技有限公司 Method and kit for dectecting target nucleic acid
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US11174470B2 (en) 2019-01-04 2021-11-16 Mammoth Biosciences, Inc. Programmable nuclease improvements and compositions and methods for nucleic acid amplification and detection
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US11273442B1 (en) 2018-08-01 2022-03-15 Mammoth Biosciences, Inc. Programmable nuclease compositions and methods of use thereof
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US11584955B2 (en) 2017-07-14 2023-02-21 Shanghai Tolo Biotechnology Company Limited Application of Cas protein, method for detecting target nucleic acid molecule and kit
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Families Citing this family (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020046809A1 (en) * 2018-08-27 2020-03-05 The Regents Of The University Of California Reporter nucleic acids for type v crispr-mediated detection
CN111944879B (en) * 2019-05-16 2024-05-14 华南师范大学 Gene detection method based on CRISPR technology for non-disease diagnosis purpose
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US11851702B2 (en) 2020-03-23 2023-12-26 The Broad Institute, Inc. Rapid diagnostics
US20240052436A1 (en) 2020-06-12 2024-02-15 Sherlock Biosciences Crispr-based sars-cov-2 detection
CN111733216B (en) * 2020-06-22 2023-03-28 山东舜丰生物科技有限公司 Method for improving detection efficiency of target nucleic acid
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WO2022080343A1 (en) * 2020-10-13 2022-04-21 キヤノン株式会社 Nucleic acid detection device and nucleic acid detection method
US20240076712A1 (en) * 2021-01-08 2024-03-07 Wuhan University Compositions and methods for instant nucleic acid detection
CA3210883A1 (en) 2021-02-09 2022-08-18 Sherlock Biosciences, Inc. Nucleic acid amplification using promoter primers
CN113061650B (en) * 2021-02-19 2024-07-16 中国科学院深圳先进技术研究院 System and method for detecting pathogen nucleic acid in real time
NO347409B1 (en) * 2021-04-11 2023-10-23 Glomsroed Mikkel Ante PROCEDURE FOR THE DETECTION OF MICROORGANISMS OR GENE DEFECTS
CN113444817A (en) * 2021-05-12 2021-09-28 中国人民解放军军事科学院军事医学研究院 Bacillus anthracis detection method based on CRISPR-Cas12a system
CN113403424B (en) * 2021-05-28 2024-01-09 华南理工大学 Method and kit for rapidly detecting novel coronaviruses and mutant strains based on CRISPR/Cas12a technology
CN113337489A (en) * 2021-06-07 2021-09-03 广州美格生物科技有限公司 Novel chimera TsCas12a protein and preparation technology
CN113373264B (en) * 2021-06-10 2022-04-12 安徽医科大学 Novel coronavirus double-target rapid detection method and kit based on CRISPR/Cas system
CN113355397A (en) * 2021-06-24 2021-09-07 深圳市第二人民医院(深圳市转化医学研究院) Field effect transistor chip, kit for detecting nucleic acid and method for detecting nucleic acid content
CN113980957A (en) * 2021-07-02 2022-01-28 苏州系统医学研究所 Single-stranded DNA probe based on CRISPR/Cas12a and method for detecting target nucleic acid
CN113388691B (en) * 2021-07-23 2022-08-30 中国疾病预防控制中心传染病预防控制所 Nucleic acid detection method based on PCR amplification and CRISPR-Cas12a and application
CN115247206A (en) * 2021-10-21 2022-10-28 河北大学 Method for detecting nucleic acid marker based on ligase chain reaction and gene editing technology
WO2023087290A1 (en) * 2021-11-20 2023-05-25 Agricultural Genomics Institute At Shenzhen, Chinese Academy Of Agricultural Sciences Methods for nucleic acid detection
CN114134218B (en) * 2021-12-02 2022-10-28 中国疾病预防控制中心传染病预防控制所 Fluorescent detection method based on CRISPR-Cas12a
CN114292843B (en) * 2021-12-03 2023-07-21 中国科学院精密测量科学与技术创新研究院 CRISPR/Cas12a detection system of gene stimulant and application thereof
CN114807316B (en) * 2022-03-11 2023-02-03 北京科技大学 RNA quantitative detection method without nucleic acid amplification visualization
CN116240200A (en) * 2022-07-01 2023-06-09 中国科学院基础医学与肿瘤研究所(筹) Ultrasensitive target nucleic acid enrichment detection method based on programmable nuclease
WO2024006552A1 (en) 2022-07-01 2024-01-04 Sherlock Biosciences, Inc. Ambient temperature nucleic acid amplification and detection
CN116179511B (en) * 2023-03-10 2023-12-22 之江实验室 Application of Cpf1 protein in preparation of kit for nucleic acid detection
CN117821630A (en) * 2024-02-20 2024-04-05 江苏省家禽科学研究所 Nucleic acid detection kit and method for detecting salmonella enteritidis

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105441440A (en) * 2012-10-23 2016-03-30 基因工具股份有限公司 Composition for cleaving a target DNA comprising a guide RNA specific for the target DNA and CAS protein-encoding nucleic acid or CAS protein, and use thereof
WO2017053762A1 (en) * 2015-09-24 2017-03-30 Sigma-Aldrich Co. Llc Methods and reagents for molecular proximity detection using rna-guided nucleic acid binding proteins

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101935697B (en) * 2010-04-16 2015-11-25 中生方政生物技术有限公司 The method detected for nucleotide sequence and test kit
RU2678001C2 (en) * 2012-12-13 2019-01-22 ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи Dna detection methods for site-specific nuclease activity
WO2016123243A1 (en) * 2015-01-28 2016-08-04 The Regents Of The University Of California Methods and compositions for labeling a single-stranded target nucleic acid
US9790490B2 (en) * 2015-06-18 2017-10-17 The Broad Institute Inc. CRISPR enzymes and systems
EP3365441A1 (en) * 2015-10-22 2018-08-29 The Broad Institute Inc. Type vi-b crispr enzymes and systems
CA2947904A1 (en) * 2015-11-12 2017-05-12 Pfizer Inc. Tissue-specific genome engineering using crispr-cas9
CN107488710B (en) * 2017-07-14 2020-09-22 上海吐露港生物科技有限公司 Application of Cas protein, and detection method and kit of target nucleic acid molecule
US10253365B1 (en) * 2017-11-22 2019-04-09 The Regents Of The University Of California Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105441440A (en) * 2012-10-23 2016-03-30 基因工具股份有限公司 Composition for cleaving a target DNA comprising a guide RNA specific for the target DNA and CAS protein-encoding nucleic acid or CAS protein, and use thereof
WO2017053762A1 (en) * 2015-09-24 2017-03-30 Sigma-Aldrich Co. Llc Methods and reagents for molecular proximity detection using rna-guided nucleic acid binding proteins

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALEXANDRA EAST-SELETSKY 等: "Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection", 《NATURE》 *
JONATHAN S. GOOTENBERG 等: "Nucleic acid detection with CRISPR-Cas13a/C2c2", 《SCIENCE》 *
PU GAO 等: "Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition", 《CELL RESEARCH》 *
SHI-YUAN LI 等: "CRISPR-Cas12a-assisted nucleic acid detection", 《CELL DISCOVERY》 *
李诗渊 等: "合成生物学技术的研究进展——DNA 合成、组装与基因组编辑", 《生物工程学报》 *

Cited By (110)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019011022A1 (en) * 2017-07-14 2019-01-17 上海吐露港生物科技有限公司 Application of cas protein, method for detecting target nucleic acid molecule and kit
US11584955B2 (en) 2017-07-14 2023-02-21 Shanghai Tolo Biotechnology Company Limited Application of Cas protein, method for detecting target nucleic acid molecule and kit
WO2019104058A1 (en) 2017-11-22 2019-05-31 The Regents Of The University Of California Type v crispr/cas effector proteins for cleaving ssdnas and detecting target dnas
US11118224B2 (en) 2017-11-22 2021-09-14 The Regents Of The University Of California Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs
CN111630163A (en) * 2017-11-22 2020-09-04 加利福尼亚大学董事会 V-type CRISPR/CAS effector proteins for cleaving SSDNA and detecting target DNA
US10253365B1 (en) 2017-11-22 2019-04-09 The Regents Of The University Of California Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs
EP3714050A4 (en) * 2017-11-22 2021-08-18 The Regents of The University of California Type v crispr/cas effector proteins for cleaving ssdnas and detecting target dnas
US11447824B2 (en) 2017-11-22 2022-09-20 The Regents Of The University Of California Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs
CN108220400A (en) * 2018-01-02 2018-06-29 武汉启丰生物技术有限公司 A kind of quick detection of nucleic acids sizing technique based on CRISPR-Cas10 nucleic acid-protein compounds
CN111615561A (en) * 2018-03-15 2020-09-01 荣研化学株式会社 Oligonucleotide probe for detecting single nucleotide polymorphism and cis-trans discrimination method
US11851701B2 (en) 2018-03-15 2023-12-26 Eiken Kagaku Kabushiki Kaisha Oligonucleotide probe for detecting single nucleotide polymorphisms, and method for determining cis-trans configuration
CN111615561B (en) * 2018-03-15 2024-06-04 荣研化学株式会社 Oligonucleotide probe for detecting single nucleotide polymorphism and cis-trans discrimination method
CN108796036A (en) * 2018-04-03 2018-11-13 上海交通大学 Nucleic acid detection method based on protokaryon Argonaute albumen and its application
CN108588182A (en) * 2018-04-13 2018-09-28 中国科学院深圳先进技术研究院 Isothermal duplication and detection technique based on the substitution of CRISPR- chains
CN112941155B (en) * 2018-05-14 2023-07-14 北京艾克伦医疗科技有限公司 DNA primer pair with stem-loop structure and application thereof
CN112899350B (en) * 2018-05-14 2023-01-24 北京艾克伦医疗科技有限公司 Nucleic acid detection method and kit
CN112941155A (en) * 2018-05-14 2021-06-11 北京艾克伦医疗科技有限公司 DNA primer pair with stem-loop structure and application thereof
CN112899350A (en) * 2018-05-14 2021-06-04 北京艾克伦医疗科技有限公司 Nucleic acid detection method and kit
WO2019233385A1 (en) * 2018-06-03 2019-12-12 上海吐露港生物科技有限公司 Use of high-temperature-resistant cas protein, and method and reagent kit for detecting target nucleic acid molecule
CN112236530A (en) * 2018-06-03 2021-01-15 上海吐露港生物科技有限公司 Application of high-temperature-resistant Cas protein, and detection method and kit of target nucleic acid molecule
CN110551800A (en) * 2018-06-03 2019-12-10 上海吐露港生物科技有限公司 Application of high-temperature-resistant Cas protein, and detection method and kit of target nucleic acid molecule
JP7431453B2 (en) 2018-06-03 2024-02-15 シャンハイ トロ バイオテクノロジー カンパニー リミテッド Use of high temperature resistant Cas protein and method and kit for detecting target nucleic acid molecules
AU2019280305B2 (en) * 2018-06-03 2023-07-20 Shanghai Tolo Biotechnology Company Limited Use of high-temperature-resistant Cas protein, and method and reagent kit for detecting target nucleic acid molecule
JP2021528098A (en) * 2018-06-03 2021-10-21 シャンハイ トロ バイオテクノロジー カンパニー リミテッドＳｈａｎｇｈａｉ Ｔｏｌｏ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｃｏｍｐａｎｙ Ｌｉｍｉｔｅｄ Use of high temperature resistant Cas protein and detection method and kit of target nucleic acid molecule
US12104204B2 (en) 2018-06-03 2024-10-01 Shanghai Tolo Biotechnology Company Limited Use of high-temperature-resistant Cas protein, and method and reagent kit for detecting target nucleic acid molecule
EP3805403A4 (en) * 2018-06-03 2022-05-04 Shanghai Tolo Biotechnology Company Limited Use of high-temperature-resistant cas protein, and method and reagent kit for detecting target nucleic acid molecule
RU2800778C2 (en) * 2018-06-03 2023-07-28 Шангхаи Толо Биотехнолоджи Компани Лимитед Use of high-therm-resistant cas-protein and a method and a set of reagents for detection of a target nucleic acid molecule
CN112639121A (en) * 2018-06-26 2021-04-09 博德研究所 Amplification compositions, systems, and methods based on CRISPR double nickases
US11761029B2 (en) 2018-08-01 2023-09-19 Mammoth Biosciences, Inc. Programmable nuclease compositions and methods of use thereof
US11273442B1 (en) 2018-08-01 2022-03-15 Mammoth Biosciences, Inc. Programmable nuclease compositions and methods of use thereof
CN113227367B (en) * 2018-08-09 2023-05-12 G+Flas生命科学公司 Compositions and methods for genome engineering with CAS12A protein
CN113227367A (en) * 2018-08-09 2021-08-06 G+Flas生命科学公司 Compositions and methods for genome engineering using CAS12A protein
WO2020042901A1 (en) * 2018-08-30 2020-03-05 杭州杰毅生物技术有限公司 Crispr-cas-based isothermal method for detecting nucleic acid and kit
WO2020056924A1 (en) * 2018-09-20 2020-03-26 中国科学院动物研究所 Method for detecting nucleic acid
JP7500549B2 (en) 2018-09-20 2024-06-17 インスティテュート オブ ズーオロジー、チャイニーズ アカデミー オブ サイエンシーズ Methods for detecting nucleic acids
CN109837328A (en) * 2018-09-20 2019-06-04 中国科学院动物研究所 Nucleic acid detection method
JP2022501039A (en) * 2018-09-20 2022-01-06 インスティテュート オブ ズーオロジー、チャイニーズ アカデミー オブ サイエンシーズ How to detect nucleic acids
CN109837328B (en) * 2018-09-20 2021-07-27 中国科学院动物研究所 Nucleic acid detection method
CN111117984A (en) * 2018-11-01 2020-05-08 浙江大学 CRISPR system, method and device for visually and specifically detecting nucleic acid target
CN111154913B (en) * 2018-11-08 2023-04-18 中山大学 Primers and crRNA for EB virus DNA detection and application thereof
CN111154913A (en) * 2018-11-08 2020-05-15 中山大学 Primer and crRNA for EB virus DNA detection and application thereof
CN109251963B (en) * 2018-11-12 2022-11-18 复旦大学 Method and kit for detecting mycoplasma pollution in cell culture solution at constant temperature
CN109251963A (en) * 2018-11-12 2019-01-22 复旦大学 The method and kit of mycoplasma contamination in Constant Temperature Detection cell culture fluid
CN109666662A (en) * 2018-12-12 2019-04-23 广州普世利华科技有限公司 Application of the novel ScCas12a in terms of detection of nucleic acids
US11174470B2 (en) 2019-01-04 2021-11-16 Mammoth Biosciences, Inc. Programmable nuclease improvements and compositions and methods for nucleic acid amplification and detection
CN109628564B (en) * 2019-02-25 2022-02-01 北京市理化分析测试中心 Primer set for detecting SNP polymorphism and method for detecting SNP polymorphism by using primer set
CN109628564A (en) * 2019-02-25 2019-04-16 北京市理化分析测试中心 A method of for detecting the primer sets of SNP polymorphism and detecting SNP polymorphism using primer sets
CN109929949A (en) * 2019-03-14 2019-06-25 广州医科大学附属肿瘤医院 The detection method and application of EBV based on CRISPR-Cas12a and G tetra- serobilas-ferroheme
CN110117818A (en) * 2019-04-16 2019-08-13 天津大学 Detect the CRISPR high flux biochip of single gene mutation
CN110029194A (en) * 2019-04-24 2019-07-19 安邦（厦门）生物科技有限公司 Continuous fluorescence based on CRISPR-Cas gene editing technology monitors detection method and device
CN111855990A (en) * 2019-04-29 2020-10-30 华南师范大学 Universal colorimetric nucleic acid detection method based on CRISPR/Cas system, kit and application
CN111855990B (en) * 2019-04-29 2023-06-27 华南师范大学 CRISPR/Cas system-based universal colorimetric nucleic acid detection method, kit and application
CN110257556A (en) * 2019-04-30 2019-09-20 广州普世利华科技有限公司 A kind of kit for detecting nucleic acid of sexually transmitted disease infective pathogen
CN110195102A (en) * 2019-04-30 2019-09-03 广州普世利华科技有限公司 A kind of beta Thalassemia methods of genotyping
CN110257556B (en) * 2019-04-30 2023-04-18 广州普世利华科技有限公司 Nucleic acid detection kit for pathogenic pathogen of sexually transmitted diseases
CN110157821A (en) * 2019-05-06 2019-08-23 浙江大学 A kind of kit for Human fetal cardiomyocytes detection
CN110157821B (en) * 2019-05-06 2020-08-28 浙江大学 Kit for detecting human staphylococcus
CN110317890A (en) * 2019-06-24 2019-10-11 浙江大学 A kind of kit for enterococcus faecalis detection
CN110358848A (en) * 2019-06-24 2019-10-22 浙江大学 A kind of kit for enterococcus faecium detection
CN110241238A (en) * 2019-06-24 2019-09-17 浙江大学 A kind of kit for streptococcus pyogenes detection
CN110331219A (en) * 2019-06-24 2019-10-15 浙江大学 A kind of kit for pseudomonas aeruginosa detection
CN112301101B (en) * 2019-07-24 2024-07-19 上海吐露港生物科技有限公司 CRISPR multi-target detection method and kit thereof
CN112301101A (en) * 2019-07-24 2021-02-02 上海吐露港生物科技有限公司 CRISPR multi-target detection method and kit thereof
CN114174535A (en) * 2019-07-24 2022-03-11 上海吐露港生物科技有限公司 CRISPR multi-target detection method and kit thereof
CN110819652A (en) * 2019-08-21 2020-02-21 山东金城生物药业有限公司 Dual-function system for simultaneous gene knockout and gene expression inhibition of corynebacterium glutamicum and application
CN110894557A (en) * 2019-09-20 2020-03-20 武汉大学 CRRNA (crribonucleic acid) for detecting African swine fever virus based on CRISPR (clustered regularly interspaced short palindromic repeats) mode and kit
CN110551812B (en) * 2019-09-26 2020-08-18 暨南大学 CRISPR-Cas system for diagnosing spinal muscular atrophy and application thereof
CN110551812A (en) * 2019-09-26 2019-12-10 暨南大学 CRISPR-Cas system for diagnosing spinal muscular atrophy and application thereof
CN111007240A (en) * 2019-12-02 2020-04-14 中国医学科学院北京协和医院 Homogeneous enzyme immunoassay system based on CRISPR technology and method and application thereof
CN110878343A (en) * 2019-12-03 2020-03-13 国家卫生健康委科学技术研究所 Cpf1 kit for quickly detecting genetic deafness pathogenic gene SLC26A4 mutation and detection method thereof
CN110878343B (en) * 2019-12-03 2023-04-07 国家卫生健康委科学技术研究所 Cpf1 kit for rapidly detecting genetic deafness pathogenic gene SLC26A4 mutation and detection method thereof
CN112986551B (en) * 2019-12-11 2022-09-27 北京聚树生物科技有限公司 Method and kit for detecting concentration of target molecules in mixed system
CN112986551A (en) * 2019-12-11 2021-06-18 清华大学 Method and kit for detecting concentration of target molecules in mixed system
CN110894530A (en) * 2019-12-25 2020-03-20 武汉博杰生物医学科技有限公司 Detection kit and detection method for gene mutation of colon cancer-related molecular marker
CN111304294A (en) * 2020-01-17 2020-06-19 中国医学科学院北京协和医院 Rapid detection system and method for combination of PCDA (Poly carbonate-co-polymerase chain reaction) vesicles and CRISPR (clustered regularly interspaced short palindromic repeats)
WO2021154866A1 (en) 2020-01-27 2021-08-05 Sherlock Biosciences Improved detection assays
WO2021155775A1 (en) * 2020-02-03 2021-08-12 苏州克睿基因生物科技有限公司 Method and kit for dectecting target nucleic acid
CN111455046A (en) * 2020-03-13 2020-07-28 深圳市罗湖区人民医院 Nucleic acid detection method for mutation of ADGRG6 enhancer and kit thereof
CN111440793A (en) * 2020-03-20 2020-07-24 武汉博杰生物医学科技有限公司 Novel coronavirus nucleic acid detection kit
CN111560469A (en) * 2020-03-30 2020-08-21 广州和盛医疗科技有限公司 Primer group for detecting new coronavirus gene, CRISPR (clustered regularly interspaced short palindromic repeats) sequence combination and application of primer group
CN111257297A (en) * 2020-03-31 2020-06-09 海南大学 RNA detection method based on Cas14a enzyme
CN111363763A (en) * 2020-03-31 2020-07-03 海南大学 Method for activating Cas14a enzyme collateral cleavage effect by RNA
CN111549177A (en) * 2020-04-27 2020-08-18 广州再生医学与健康广东省实验室 gRNA and kit for detecting SARS-CoV-2
CN111719015A (en) * 2020-05-21 2020-09-29 昆明医科大学第一附属医院 Human immunodeficiency virus HIV-1 detection kit
CN111662968A (en) * 2020-06-23 2020-09-15 南方科技大学 Detection method of PAM-sequence-free DNA based on CRISPR and application thereof
CN112795624A (en) * 2020-07-17 2021-05-14 山东舜丰生物科技有限公司 Method for detecting target nucleic acid using nucleic acid detector containing abasic spacer
CN111909987A (en) * 2020-07-31 2020-11-10 浙江科途医学科技有限公司 Reagent composition, kit and detection system for KIF5B-RET fusion gene detection
CN112251494B (en) * 2020-10-09 2022-09-02 湖南大学 Nucleic acid detection method based on CRISPR-Cas autocatalytic amplification network and application thereof
CN112251494A (en) * 2020-10-09 2021-01-22 湖南大学 Nucleic acid detection method based on CRISPR-Cas autocatalytic amplification network and application thereof
CN112301162A (en) * 2020-10-22 2021-02-02 清华-伯克利深圳学院筹备办公室 Virus nucleic acid detection method for simultaneously detecting DNA virus and RNA virus
CN112301162B (en) * 2020-10-22 2023-09-22 清华-伯克利深圳学院筹备办公室 Virus nucleic acid detection method for simultaneously detecting DNA virus and RNA virus
CN114561450A (en) * 2020-11-27 2022-05-31 佳能株式会社 Method and kit for detecting target nucleic acid
CN112695073A (en) * 2020-12-30 2021-04-23 上海理工大学 CRISPR/Cas12a one-step nucleic acid detection method
CN112831544A (en) * 2020-12-31 2021-05-25 华南农业大学 Biological detection method and biological detection device based on CRISPR/Cas12a system
CN113046477A (en) * 2021-02-26 2021-06-29 广州医科大学附属第一医院（广州呼吸中心） Specific crRNA for identifying D614G mutation site, system, kit and application
CN114807431A (en) * 2021-04-15 2022-07-29 华中农业大学 Improved nucleic acid visual detection technology based on CRISPR system mediation and application thereof
CN113324956A (en) * 2021-04-22 2021-08-31 上海市肿瘤研究所 CRISPR technology and framework nucleic acid coupling-based modular detection platform, construction method and application thereof
CN112852980B (en) * 2021-04-23 2021-11-26 上海海关动植物与食品检验检疫技术中心 Detection method for detecting nucleic acid of Atlantic salmon based on CRISPR fluorescence method and detection kit thereof
CN112852980A (en) * 2021-04-23 2021-05-28 上海海关动植物与食品检验检疫技术中心 Detection method for detecting nucleic acid of Atlantic salmon based on CRISPR fluorescence method and detection kit thereof
CN113686934A (en) * 2021-08-13 2021-11-23 广东海洋大学 CRISPR/Cas12a-RCA electrochemical sensor detection system and application thereof
CN113999921B (en) * 2021-11-23 2023-06-27 中国医学科学院输血研究所 Method and kit for rapidly and visually detecting shigella flexneri
CN113999921A (en) * 2021-11-23 2022-02-01 中国医学科学院输血研究所 Method and kit for rapidly and visually detecting Shigella flexneri
CN114592043A (en) * 2022-03-23 2022-06-07 北京盛因生物科技有限公司 Isothermal nucleic acid detection enzyme composition, kit, application and detection method thereof
CN114634972A (en) * 2022-05-19 2022-06-17 舜丰生物科技(海南)有限公司 Method for detecting nucleic acid by using Cas enzyme
CN115838831A (en) * 2022-08-29 2023-03-24 深圳市海微生物科技有限公司 Application of FnCas12a mutant in nucleic acid detection and nucleic acid detection method
CN116297376A (en) * 2023-04-25 2023-06-23 中国科学院过程工程研究所 Photoelectric dual-mode biological probe and application thereof
CN116297376B (en) * 2023-04-25 2023-08-29 中国科学院过程工程研究所 Photoelectric dual-mode biological probe and application thereof
CN116574848A (en) * 2023-06-20 2023-08-11 中国农业科学院兰州兽医研究所 Primer and detection method for detecting porcine circovirus type 2 based on LAMP-CRISPRCas12a
CN116656784B (en) * 2023-07-26 2023-10-20 南京鸿明生物科技有限公司 Titer measurement method of amplification-free adeno-associated virus genome
CN116656784A (en) * 2023-07-26 2023-08-29 南京鸿明生物科技有限公司 Titer measurement method of amplification-free adeno-associated virus genome

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